ABSTRACT A complete diallel cross between two geographically
distant pearl oyster (Pinctada martensii) populations, an Indian
cultured population (I), and a Chinese cultured population (C) was
carried out, and the resulting progenies (II, CC, IC, and CI) were
cultured and studied for more than 700 days. Shell height and total
weight were measured monthly, and shell thickness was measured in the
middle and at the end of the experiment. The results reveal that II grew
fastest whereas CC grew the slowest. The growth rate of reciprocal
crosses CI and IC exhibited no statistically significant difference,
with both appearing to be intermediate between the parental species, but
superior to the mid parent values. The morphological traits of parents
were inherited differently by the two reciprocal crosses. The traits of
large size and relative thinness of shell from the Indian population
were largely transmitted to CI, whereas relative small size and
increased shell thickness of the Chinese population were mostly
inherited by IC. The two parental stocks, the Indian population and the
Chinese population, were unsuitable for commercial production because of
a relatively thin shell and slow growth, respectively, but the
reciprocal crosses combined desirable traits of the parents and
exhibited considerable potential for commercial production and pearl
culture.

The marine pearl oyster Pinctada martensii is widely distributed
throughout the equatorial zone, from the western Pacific Ocean (Korea
and southern China), Australia, and the Indian Ocean to the Red Sea and
the Persian Gulf, with Lessepsian migrants through the Suez Canal into
the Mediterranean (Gervis & Sims 1992). It is commercially cultured
in Japan, China, India, and Vietnam (Southgate & Lucas 2008) for the
production of round pearls, popularly known as "Akoya pearls."
These pearls are 5-10 mm in diameter and are most widely used in
necklaces, especially the 14-16-inch choker and the 17-19-inch princess
(Kripa et al. 2007). In China, P. martensii oysters were introduced from
Japan to China in the 1960s and have been cultured for more than 40 y in
3 southern coastal provinces: Guangdong, Guangxi, and Hainan. The pearls
produced by this species comprise more than 90% of the country's
marine pearl production (Guo et al. 1999). In the past 2 decades, the
production of Akoya pearls in China has increased dramatically. By the
end of the 1980s and 1990s, its annual production was estimated to be
more than 2 t and 20 t, respectively (Wang et al. 2007), and it reached
29.3 t in 2003 (Chen & Li 2007).

However since P. martensii oysters were first introduced from Japan
more than 40 y ago, the only breeding method used by farmers has been
selection, conducted in an informal manner with minimal assessment of
efficacy. Because of the limited number of parents used for mating,
genetic variation has been lost and inbreeding depression has arisen
(Wang et al. 2003b, Deng et al. 2006, He et al. 2006), resulting in a
decrease in oyster size and growth rate, which has directly affected the
size of the nucleus and the value of the pearl. Therefore, P. martensii
in China is in urgent need of genetic improvement to cultivate
fast-growing and larger species.

In this study, a cross between two geographically distant
populations an Indian population and a Chinese population--of P.
martensii was conducted to determine whether there was a significant
improvement in growth of reciprocal crosses from pure populations. In
pearl production, shell height (SH) and shell thickness (ST) are the
chief criteria in deciding whether oysters are appropriate for
implantation, and the size of the nuclei that may be seeded. In China,
oysters are considered mature enough for implantation when SH is 60 mm
and they can be inserted with a nuclei of 6.5 mm. If the nuclei are
larger, oysters should be larger, too (Gu et al. 2009). In addition to
SH, ST is another criterion, because oysters with thin shells are unfit
for implantation of large nuclei (Kripa et al. 2007). In this
experiment, Indian and Chinese populations differ in morphology and
growth rate, and are complementary in SH and ST. The Indian cultured
population is large and fast growing; however, its shells are rather
thin and fragile, and easy to crack during implantation, thus it is
impossible to produce pearls larger than 5 mm (Kripa et al. 2007). In
comparison, as observed by local farmers, the local Chinese population
has thick, tough shells, but it is small and has a low growth rate, and
hence is unfit for the implantation of large nuclei. The objective of
this study was to explore the possibility of producing desirable traits
such as faster growth and greater ST in the reciprocal crosses compared
with intrapopulation crosses under identical culture systems.

MATERIALS AND METHODS

Study Site

The study was undertaken in Li'an Lagoon
(18[degrees]24'1-18[degrees]27, N,
110[degrees]02'-110[degrees]04' E), Hainan Island, South
China. This lagoon, stretching 4 km north to south and 2.8 km east to
west, is linked with the ocean by a narrow branch 60 m wide in the
southeast. The mean water temperature is around 26[degrees]C, ranging
from 19-30[degrees]C (Gu et al. 2009); salinity is 31-34%0, with a water
depth of 8.5 m and an average flow rate of 3 cm/sec at the farming
location.

Breeding Design

In this experiment, the two parental stocks the Indian and Chinese
populations produced 4 crosses: 2 intrapopulation crosses (II and CC; I,
Indian; C, Chinese) and 2 reciprocal crosses (IC and CI). For
consistency, when referring to the between-population crosses, the
maternal species is named first.

The parental stocks of the Indian and Chinese populations were
collected from a commercial farm in Li'an Lagoon, Hainan Island,
and reared under identical conditions. The Indian group is the [F.sub.2]
offspring of 200 oysters introduced from the Bay of Bengal along the
southeast coast of India in 2002 to Hainan and reared in Li'an
Lagoon for experiment. The Chinese Sanya group is cultured locally,
whose ancestors are the wild pearl oysters distributed in the coastal
waters of Sanya Bay, and have been reared commercially in Li'an
Lagoon for more than 5 generations for pearl production.

On March 15, 2003, 80 2.5-y-old oysters from each population were
collected and transported to the laboratory at Li'an, where they
were cleaned and immersed in 5 ppm potassium permanganate for 5 min for
disinfection (Wang et al. 2003a). Then, they were measured and further
divided into males and females by pricking the gonad with a needle and
observing the reproductive cells under a microscope. Last, 30 female and
male oysters were chosen from each population as parents for breeding.
The parameters of the parents are shown in Table 1.

The mating design is shown in Figure I. Males and females of one
population were separately placed in two aerated tanks, each containing
200 L filtered seawater. All oysters were injected with 0.1 mL of 0.02
mM serotonin (5-droxytryptamine) (Sigma) into the adductor muscle to
induce spawning (Zhang et al. 2007). Males were injected first, followed
by females, because eggs have a shorter lifespan than sperm, and if eggs
are exposed to the air for a long time, their quality may degrade.
Around half an hour after injection, oysters began to spawn, and more
than 80% of males and females participated in the production of
offspring. After spawning, the sperm or the eggs in each tank were
stirred and split into two halves. Then eggs were fertilized as shown in
Figure 1, and 4 crosses were produced: II, CC, IC, and CI.

Larvae Rearing and Culture of Spat and Adults

The fertilized eggs were collected on a 38-[micro]m mesh sieve and
reared in 1,000-L aerated fiberglass tanks for hatching. Four to 5 h
after fertilization, when the fertilized eggs developed into trochophore
larvae, aeration was stopped and the larvae were collected from the
upper layer of the tanks and transferred to 30,000-L cement pits, where
the density was maintained at 4 larvae/mL. When the eye spots appeared,
polyethylene rope-type collectors were deployed into the larval tanks
(Gervis & Sims 1992). After that, the rearing of spat was carried
out using standard practices (Gu et al. 2009). When spat reached a
2.5-cm SH, they were transferred into circle nets with 2.0-cm mesh and
were reared at a density of 200-300 spat per net. Thereafter, net
changing, shell cleaning, and density adjustment were conducted
regularly in accordance with normal commercial practice (Wada &
Komaru 1991).

Because all oysters were farmed in natural waters, their growth
might be easily affected by environmental factors--in particular,
temporal differences in food abundance. To minimize such environmental
effects, all oysters of the four crosses were arranged in a line in the
same area normal to the direction of tidal currents. Specifically, the
tidal currents in Li'an Lagoon flow from south to north, and the
oyster nets of the 4 crosses were deployed along a 180-m east-west
long-line, so that the oysters could obtain equal amounts of food
brought by tidal currents.

Measurement and Statistical Analysis

The measurement of SH ([+ or -] 0.01 mm) was conducted from May
2003 to March 2005, whereas the measurement of total weight (TW; [+ or
-] 0.01 g) began 1 mo later, because in May 2003, oysters were not heavy
enough to allow for a weight measurement. A total of 100 oysters from
each cross were randomly collected, cleaned, and then measured (Hwang et
al. 2007). In addition to SH and TW, in January 2004 and March 2005, ST
was also measured, and relative thickness was calculated by ST divided
by SH.

[FIGURE 1 OMITTED]

Oysters are suitable for nucleus implantation when SH reaches 60 mm
(Gu et al. 2009). Therefore, the numbers of oysters in the 4 groups with
an SH more than 60 mm were calculated at different times.

One-way analysis of variance with a posteriori Tukey HSD tests were
used to test the null hypothesis that the means of SH, ST, TW, or ST/SH
in the 4 groups are equal at [alpha] = 0.01 (PASW Statistics, 2009).

RESULTS

Growth

The growth trends for SH and TW of four groups are shown in Figure
2B. The reciprocal crosses CI and IC were intermediate to their parental
species, and comparatively much closer to their fast-growing parental
Indian population. Both SH and TW of the reciprocal crosses were
significantly larger than those of the slow-growing CC, but
significantly smaller than the fast-growing II cross (P < 0.01) under
identical rearing conditions (Tables 2 and 3). The two reciprocal
crosses did not show a statistically significant difference in SH or TW
(P > 0.05; Tables 2 and 3), except that on days 53 and 87, IC had a
significantly larger SH than CI (P > 0.05).

Morphological Features

Four groups differed in morphology as well. Mean values of ST and
ST/SH on days 300 and 727 (the end of the experiment) are summarized in
Tables 2 and 3. On day 300, II had the largest ST but the smallest
ST/SH, whereas CC had the smallest ST, but significantly larger ST/SH
(Table 2). Therefore, in morphology, II was large but relatively thin,
whereas CC was small but relatively thick. Reciprocal crosses IC and CI
showed different traits, and on day 727, IC had a significantly higher
ST/SH than CI (Table 3). The final results show that the ST/SH of the 4
crosses differed significantly (P < 0.01) from each other, but the
values of the two reciprocal crosses were comparatively much closer to
the thick parental Chinese population.

Proportion and Age of Pearl Oysters Fit for Implantation

Figure 3 shows the proportion and age of oysters (SH [greater than
or equal to] 60 ram) large enough for implantation of the 4 groups.
Oysters fit for implantation were observed to appear about 290 clays
after fertilization. At the end of the experiment, II, CI, and IC had
approximately 100% of oysters ready for implantation, but CC had only
45%. Of the 4 groups, the II group generally had the largest proportion
of oysters suitable for implantation, followed by CI, IC, and CC. For
example, on day 543, the percentages of II, CI, IC and CC were 80.6%,
62.5%, 40.5%, and 6% respectively.

In comparison, few studies have been conducted on the pearl oyster
P. martensii except those by Wada and Komaru (1994) and Wang et al.
(2003b, 2004). Wada and Komaru (1994) crossed pearl oysters of different
shell coloration and found hybrid crosses superior to inbred crosses in
survival and growth rates. Wang et al. (2003b, 2004) carried out crosses
of three geographically different populations and found no significant
improvement in growth. A possible reason for the lack of differences
between these crosses might be a lack of genetic traits, because the
three populations were collected from Guangdong, Guangxi, and Hainan,
which are all located in the south of China. In the experiment by Wang
et al. (2003b, 2004), after 18 months of rearing, the three
intrapopulation crosses were almost the same in SH (47-48 cm), ST (19-20
cm), and TW (13-14 g). Not surprisingly, the 3 between-population
crosses resembled each other in the 3 growth parameters. However, in the
current study, the two parental stocks differed in geography,
morphology, and genetic traits. The Indian population had a
significantly larger SH, ST, and TW, and a significantly smaller
relative ST/SH than the Chinese population (Table 1).

Hou et al. (2008) found high differentiation between the 2 parental
populations (mean FST, 0.486), and a moderate level of genetic
diversity. They also reported that the traits of the Indian and Chinese
populations were highly complementary, and expected to show heterosis in
between-population crosses. The results of the current study reveal that
reciprocal crosses had superior performance over the mid-parent values
and exhibited many desirable traits. Similar results were observed in
scallop (Cruz & Ibarra 1997) and Pacific oyster (Soletchnik et al.
2002).

The two reciprocal crosses in the current study were variable and
exhibited different features in growth and morphology. IC exhibited
significantly faster SH than CI in the first 3 months, almost the same
in the fourth month, was surpassed by CI in the fifth month, and
maintained the same trend until the end of the experiment (Fig. 2A).
Probably this variation was caused by maternal effects, which for most
part are produced by both genotype and nongenetic factors of female
breeders (Pirchner 1983). In the current study, the maternal effects
probably lasted for 3 mo, because the SH results of the first 3 months
suggested that the eggs from the Indian population resulted in larger
oysters than the eggs from the Chinese population. However, reports on
maternal effects from other sources diverge considerably. Most studies
found that maternal effects are expected to have the greatest impact on
traits during the early stages of development, especially at the larvae
stage (Newkirk et al. 1977, Mallet & Haley 1984, Cruz & Ibarra
1997, Zhang et al. 2007). Bivalves are subject to a maternal effect
during the first stages of their life (Pirchner 1983), because egg
reserves are the principle determinant for early growth and survival
(Cragg & Crisp 1991). Maternal effects usually fade away at older
ages, and are sometimes balanced by compensatory growth in one of the
reciprocal crosses (Cruz & Ibarra 1997). On the other hand, some
studies also proved that the maternal effects extended to the juvenile
stage or adulthood, as is the case in the current study, in which
maternal effects lasted for about 2 mo. Soletchnik et al. (2002)
reported that hybrids of cupped oysters had clear maternal effects for
growth and reproductive characteristics by the end of the 17-mo
experiment. In hybrids of Chinook salmon (Bryden et al. 2004), maternal
effects were significant in the first and second year and then declined,
but lasted to the third and fourth year. Bentsen et al. (1998) observed
small but significant maternal effects at harvest of a diallel cross of
tilapia strains. Therefore, persistence of maternal effects into the
juvenile stage and adulthood is worthy of further investigation.

[FIGURE 3 OMITTED]

The purpose of crossbreeding is to achieve genetic improvement by
retaining the favorable traits and minimizing the weakness of parental
species. To be exact, in this study, the two parental populations varied
in morphology and growth performance. The Indian population is large and
fast growing with a relatively thin shell, whereas the Chinese
population has a relatively thick shell but is small and slow growing.
Both species are deficient in terms of appropriateness for nucleus
implantation. The thin, fragile shell of the Indian cultured population
complicates implantation and discourages attempts to produce pearls
larger than 5 mm (Kripa et al. 2007), whereas the long culture period
and small size of the Chinese population increases the costs of
commercial production. Interestingly, through crossbreeding, the
desirable characteristics--such as large size and fast growth of the
Indian population, and large relative thickness of the Chinese
population were inherited by the reciprocal crosses, and undesirable
traits were reduced. Specifically, CI carried more features of the
Indian group and gained a fast growth rate, and its ST/SH was also
improved. On the contrary, IC was more similar to CC in ST, but superior
to it in growth rate. Hence, both reciprocal crosses have considerable
potential for commercial production and pearl culture.

ACKNOWLEDGMENTS

This study was supported by the National Basic Research Program of
China (973 Program) (2010CB126405 and 2009CB126005); the Natural Science
Foundation of Hainan (2010CB126405 and 2009CB126005); the Key Laboratory
of Tropic Biological Resources; MOE, and Hainan Key Laboratory of
Tropical Hydrobiological Technology.

Chang, Y., X. Liu, J. Xiang, L. Song & X. Liu. 2006.
Hybridization effects of the different geographic population of Chlamys
farreri III. The yearlong (1-2 years old) growth and development of
Chinese population and Russian population and their reciprocal crosses.
Acta Oceanol. Sin. 28:114-120. (in Chinese, with English abstract).